1. Field of the Invention
The present invention is related to a novel xylanase composition and a method for its production. Specifically, the invention is related to a purified xylanase composition derived from Acidothermus sp., and particularly Acidothermus cellulolyticus, and the use of that enzyme in bleaching pulp and paper and treating feed compositions.
2. State of the Art
Xylanases are known to be produced by a number of different microorganisms. Several different xylanolytic enzymes are generally produced by a microorganism, each of the xylanases acting to attack different bonds in the wood complex. Attempts to use enzymes derived from both fungal and bacterial sources in industrial processes, e.g., for enhancing delignification and brightening while lowering or eliminating the use of chlorine in the bleaching of lignocellulosic pulp in the paper industry or for improving the value of animal feed have been described in the literature.
Xylanases, e.g., endo-.beta.-xylanases (EC 3.2.1.8), which hydrolyze the xylan backbone chain, have been studied for their use in bleaching lignocellulosic material. For example, in U.S. Pat. No. 5,179,021, the combination of xylanase and oxygen treatment in the bleaching of pulp is disclosed as being particularly useful. In PCT Application Publication No. WO 92/03541, a method of dissolving hemicellulose with hemicellulases derived from the fungus Trichoderma reesei is disclosed. The search for xylanases, however, has focused on thermophilic and alkalophilic xylanases which are useful under pulp bleaching conditions utilizing high temperatures and alkali. However, the use of oxygen or ozone bleaching generally occurs at a lower pH. Accordingly, it would be advantageous to discover a low pH xylanase which has significant activity at high temperatures.
Recently, several thermophilic xylanases from fungal and bacterial microorganisms have been identified. For example, a thermophilic xylanase has been isolated from Actinomadura reclassified as Microtetraspora having an optimal pH of 6.0-7.0 and temperature range of 70-80.degree. C. (Holtz, C. et al Antonie van Leewenhoek 59:1-7, 1991). EP 473 545 discloses that the bacterial strain Thermomonospora fusca produces thermostable xylanases active at temperatures 10-90.degree. C., preferably, 50-80.degree. C. over a wide pH range, i.e., from about 5-10, with the more preferred range between 6.6-9.5. In addition, WO92/18612 discloses a xylanase enzyme derived from the genus, Dictyoglomus, having activity over a broad pH range (5.0-9.0) and thermostability at temperatures ranging from 60-90.degree. C. The thermophilic cellulolytic bacteria Acidothermus cellulolyticus is described in Mohagheghi et al., Int. J. Systematic Bact., vol. 36, no. 3, pp. 435-443 (1986), and the production of cellulase is described in Shiang et al., Appl. Microb. Biotech., vol. 34, pp. 591-597 (1991). However, neither reference describes a purified xylanase which may be useful at low pH and high temperature.
Xylanases have also been useful in animal feeds to enable animals to digest the feeds more efficiently. One result of adding xylanase to feed is an improvement in the Feed Conversion Ratio (FCR) of a feed without increasing its cost per unit weight. The FCR of a feed is the ratio of the amount of feed consumed relative to the weight gain of the animal. A low FCR indicates that a given amount of feed results in a growing animal gaining proportionately more weight. This means that the animal is able to utilise the feed more efficiently. One way in which the FCR can be reduced is to improve its digestibility by an animal thereby increasing the nutritional benefit which the animal can derive from it.
However, there are various constraints on the digestibility of the nutritional components of a feed such as its starch, fat, protein and amino acid content. These constraints include:
(i) the viscosity of materials present in the animal's gut. Such viscosity is due, at least in part, to soluble non-starch polysaccharides such as mixed-linked .beta.-glucans and arabinoxylans; PA1 (ii) entrapment of nutrients within the cell walls of the feed, particularly those of the aleurone layer in cereals. Such entrapment is caused by the high levels of non-starch polysaccharides in the cell walls of cereals which are relatively resistant to break-down by the animal's digestive system. This prevents the nutrients entrapped within the cells from being nutritionally available to the animal; and PA1 (iii) a deficiency in endogenous enzyme activity, both of the animal and of the gut microbial population particularly in a young animal.
The above problems which interfere with digestibility are particularly noticeable in the case of cereal-based diets, such as those having a high wheat content.
Due to the problem of poor digestibility of nutrients from the feed, it is normally necessary to formulate feeds to contain higher levels of energy and protein providing materials in order to meet the nutritional demands of animals.
There is now a substantial body of evidence showing that incorporating certain (supplementary) enzymes in cereal-based animal feeds can be advantageous in reducing the viscosity of material present in the animal's gut. This reduction can be achieved by enzymes such as xylanases which hydrolyse soluble xylans thereby reducing digesta viscosity which is an important constraint on the process of digestion.
The xylanases which are added as supplements must be stable and active at the pH and temperature conditions found within the gastrointestinal (GI) tract of the target animal. If they are not stable and active when exposed to such in vivo conditions, then they will not be able to reduce digesta viscosity to any significant extent. It is presently known to include xylanases as a supplement in an animal feed derived from fungi such as Trichoderma longibrachiatum, Aspergillus niger and Humicola insolens. Bedford and Classen (The Journal of Nutrition, vol. 122, pp 560-569) disclose that there is a significant correlation between digesta viscosity measured in vivo in the case of broiler chickens and bodyweight gain and FCR values. In the case of wheat and rye-based diets fed to poultry, it was shown that as much as 70-80% of the variations in the weight gain and FCR are based upon differences in intestinal viscosity alone. This highlights the importance of digesta viscosity in cereal-based feeds containing high levels of soluble arabinoxylans. As digesta viscosity increases, it reduces the digestibility of all nutrients by interfering with the diffusion of pancreatic enzymes, substrates and the end products of the digestion process.
However, the use of enzyme supplements, such as xylanase, in animal feed is complicated by the processing requirements for grain supplements. Often, such enzyme supplements are obtained by impregnating the enzyme onto a physiologically acceptable carrier, such as a cereal. The impregnated carrier is mixed with the other components of the feed and then pressed into cubes or pellets for feeding directly to animals. The processes which have been developed make use of relatively high temperatures. This is firstly to improve the efficiency of the manufacturing process and secondly to produce feeds which are free from harmful bacteria, particularly Salmonella. In addition, the use of high temperatures improves the quality and durability of the resulting cubes and pellets, increases the range of ingredients which can be efficiently handled and also increases the level of liquid ingredients, such as fat and molasses, which can be incorporated into the feed.
Processing techniques for feed components currently employ relatively high temperatures for a relatively long period. Further, the mixture is subjected to relatively high pressures during pelleting to increase the durability of the cubes or pellets formed. One of the processing methods which has been developed to improve the nutritional properties of the feed is steam pelleting. This method includes the step of treating the compounded feed with steam to increase its temperature and moisture content. This step is termed conditioning. Conditioning lasts from a few seconds up to several minutes depending on the type and formulation of the feed. The temperature in the conditioner may rise to 100.degree. C. Afterwards, the feed is passed through a pelleting die which causes a rapid increase in its temperature due to friction.
Recently, a new device for pretreatment or conditioning of feeds has been introduced called an expander. This device allows sustained conditioning under pressure followed by pelleting. According to this technique, various feed components which have previously been subjected to steam-conditioning are fed into a compression screw into which more steam is injected, and the mass is then subjected to increasing pressure and shear action and then forced through a variable exit gap. The compressed product, after reduction in particle size, is fed into a standard pelleting press. The dwell time of the feed components in the expander is about 5-20 seconds, and the temperature reached may be as high as 145.degree. C. A compression pressure of about 3.5 MPa is reached, but the build-up of both temperature and pressure is very quick and both fall rapidly as the product is expelled through the exit gap. The use of expanders is advantageous because they effectively eliminate harmful bacteria, particularly Salmonella. Furthermore, it is possible to include relatively high levels of fat and other liquid ingredients in the mixture prior to pelleting. In addition, the cooking and pressure/shear action results in greater starch gelatinisation.
Unfortunately, the high temperature and high pressure processing conditions characteristic of the expander and pelleting technology, particularly when applied in the moist conditions normally encountered during pelleting, are potentially destructive to certain feed components. This is particularly true of any enzymes, including xylanases, which are present. Thus, the prior art enzymes have generally had the problem that they are not sufficiently stable under the processing conditions of commercial pelleting operations to allow economical use of such pelleting techniques.
Accordingly, even though partial solutions to the problem of enzyme stability during feed processing are available, none of them solves the problem in a totally effective manner.